This invention relates to a method for identifying inhibitors of protein-advanced glycation end product (xe2x80x9cProtein-AGExe2x80x9d hereafter) formation. The methodology is useful in determining substances of interest in impacting pathological conditions with which protein-AGE formation is associated, such as diabetes, atherosclerosis, chronic neurodegenerative diseases, such as Alzheimer""s disease, skin photoaging, and other degenerative diseases characteristic of the aging process.
Glycation, as used herein, is a non-enzymatic, posttranslational modification of proteins by reducing sugars and other reactive carbonyl species, which adversely affect protein function. Tissue deterioration and aging have been widely associated with accumulation of damage from chemical processes induced by glycation, as well as oxidative stress and UV irradiation. The accumulation in long lived proteins of glycation products and AGE products derived from glycation has been implicated in a number of age-related diseases including long term diabetic complications (Thorpe, et al., Drugs Aging 9: 69-77 (1996)), atherosclerosis (Ruderman, et al., FASEB J. 6:2905-2914 (1993); erratum FASEB J. 7(1):237 (1993)), Alzheimer""s disease (Vitek, et al., Proc. Natl. Acad. Sci. USA 91:4766-4770 (1994)), skin photo aging (Mizutani, et al., J.Invest. Dermatol 797-802 (1997) and in the general pathology of the aging process (Frye, et al., J. Biol Chem 273:18714-18719 (1998)). Long term diabetic complications from hyperglycemia eventually cause serious and life threatening pathologies such as end-stage renal disease (Baynes, et al., Diabetes 40:405-412 (1991)). Irreversible microvascular and macrovascular complications including retinopathy, neuropathy, nephropathy, atherosclerosis, and cerebrovascular disease all have been linked mechanistically to the formation of protein-AGE in connective tissue, especially on collagen, and matrix protein components. Moreover, similar events occurring at a slower rate seem to be of equal relevance for the normal aging process. (Thorpe, et al., supra).
Glycation and subsequent protein-AGE formation plays a central role in cellular carbonyl stress and glucose toxicity. Administering the glycation inhibitor aminoguanidine effectively suppresses secondary complications in rodents with experimental diabetes (Edelstein, et al., Diabetologica 35:96-97 (1992)); however, aminoguanidine is a hydrazine derivative that shows systemic toxicity upon long-term administration, since it is a potent inhibitor of catalase (Ou, et al., Biochem Phamacol 46:1139-1144 (1993)) and inducible nitric oxide synthase (Okuda, et al., J. Neuroimmunol 81:201-210 (1998)). The toxicity profile of aminoguanidine makes it unlikely that it will be used clinically. Therefore, an urgent clinical need exists for the identification and characterization of new compounds that effectively inhibit glycation and its associated pathological consequences. A high throughput screening assay that could be applied to large combinatorial compound libraries would likely lead to the identification of new glycation inhibitors.
Reactive oxygen species (xe2x80x9cROSxe2x80x9d), and reactive carbonyl species (xe2x80x9cRCSxe2x80x9d), especially dicarbonyl compounds, are key mediators of cellular damage caused by oxidative stress, glycation and UV radiation. The origin of cellular carbonyl stress as a result of glycation, lipid peroxidation, sugar autooxidation and metabolism can be seen in, e.g., FIG. 1. Oxygen dependent and independent pathways lead to the formation of various reactive carbonyl species, including 2-dicarbonyls like methylglyoxal and glyoxal, as key intermediates for the accumulation of protein damage by AGE formation. Carbonyl stress additionally originates from the metabolic generation of methylglyoxal. Briefly, early glycation products are derived from the reaction of a reducing sugar with protein amino groups (lysine and arginine) to generate aldimines (Schiff base adducts) that can undergo the Amadori rearrangement to form ketoamine adducts (Hodge, et al., J. Am. Chem Soc. 75:316-322 (1953)). Protein-AGE are generated from early glycation products by both oxidative and non-oxidative pathways in which a variety of reactive dicarbonyl compounds such as glyoxal, methylglyoxal, and 3-deoxyosones are suggested intermediates (Thornalley, et al., Biochem J. 344:109-116 (1999)).
Protein-AGE include protein N-(carboxymethyl)lysine residues (CML) (Ahmed, et al., J. Biol. Chem 261:4889-4894 (1986)), and a heterogeneous group of complex modifications such as pentosidine (Sell, et al., J. Biol. Chem. 264:21597-21602 (1989)) that are characterized by their high fluorescence and ability to cause protein-protein cross-links. Accumulation of AGE-specific fluorescence (ex. 370 nm; em. 440 nm) is a general measure of overall protein damage and it is a widely used tool of glycation research in vitro and in vivo. In some cases, reactive dicarbonyl compounds may form by auto-oxidation of the sugar itself without requiring glycation, and the presence of trace amounts of transition metal ions (Fe, Cu) has been implicated in the formation of dicarbonyl compounds and reactive oxygen species such as hydrogen peroxide (Elgawish, et al., J. Biol. Chem 271:12964-12971 (1996)). Amino acids other than lysine and arginine are also modified by glycoxidation. For example, surface exposed methionine residues in proteins are very sensitive to protein oxidation (Hall, et al., Biochem. Biophys. Acta 1121:325-330 (1992)).
Due to its abundance, glucose is assumed to be a major source of glycation and protein-AGE formation in extracellular proteins in vivo; however, glucose is only a weak glycation agent and the chemical reaction with proteins under physiological conditions occurs only over months and years (Higgins, et al., J. Biol. Chem 256:5204-5208 (1981)). In contrast to glucose, the more reactive pentoses have been implicated as sugar sources for the glycoxidation of intracellular proteins, because they are much more efficient precursors for the formation of fluorescent AGE such as pentosidine (Sell, et al., supra). An abundant cellular pentose is ADP-ribose, which is generated from NAD by multiple metabolic pathways (Cervantes-Laurean, et al., Biochemistry 32:1528-1534 (1993); Jacobson, et al., Mol. Cell Biochem. 138:207-212 (1994)). Earlier studies have focused on a pathway that involves the intranuclear generation of ADP-ribose (ADPR). Research has demonstrated that the cell nucleus is a likely site for glycation in vivo by ADP-ribose. Oxidative stress and other conditions that cause DNA strand breaks stimulate the synthesis of nuclear polymers of ADP-ribose, which are rapidly turned over generating ADP-ribose in close proximity to the long lived histones rich in lysine and arginine residues (Cervantes-Laurean, et al., supra.
In addition to the above referenced items, tissue deterioration and aging have been widely associated with accumulation of damage from chemical processes induced by oxidative stress, glycation, and UV-irradiation. Halliwell, et al., Free Radicals in Biology and Medicine (Clarendon Press, Oxford, 1989). Berlett, et al., J. Biol Chem 272:20313-20316 (1997). All of these are potent inducers of Reactive Oxygen Species (xe2x80x9cROSxe2x80x9d) and Reactive Carbonyl Species (xe2x80x9cRCSxe2x80x9d).(Anderson et al., J. Chem, Invest. 104:103-113 (1999)), which are key intermediates of accumulative protein damage during general aging and several pathological conditions, e.g. chronic inflammatory diseases (Dimon-Gadal, et al., J. Invest. Dermatol 114:984-989 (2000)); psoriasis, and diabetes. Brownlee, et al., Ann. Rev. Med 46:223-234 (1995); Brinkmann, et al., J. Biol Chem 273:18714-18719 (1998)). RCS as reactive intermediates of cellular carbonyl stress originate from a multitude of mechanistically related pathways, like glycation (Thornalley, et al., BioChemJ 344:109-116 (1999), sugar autoxidation (Wolffi, et al., Prog. Clin. Biol. Res 304:259-75 (1989), lipid peroxidation (Fu, et al., J. Biol Chem 271:9982-64996), and UV-photodamage (Mizutani, et al., J. Invest. Dermatol 108:797-802 (1997)). Glycation, a spontaneous amino-carbonyl reaction between reducing sugars and long-lived proteins is a major source of RCS production leading to cellular carbonyl stress. Reactive xcex1-dicarbonyl intermediates, such as glyoxal, methylglyoxal, and 3-deoxyosones, are generated by both oxidative (glycoxidative) and non-oxidative reaction pathways of glycation. The complex reaction sequence is initiated by the reversible formation of a Schiff base, which undergoes an Amadori rearrangement to form a relatively stable ketoamine product during early glycation. A series of further reactions involving sugar fragmentation and formation of xcex1-dicarbonyl compounds as key reactive intermediates yields stable protein-bound advanced glycation end products (AGEs) (Thornalley, et al., supra; Glomb, et al., J. Biol. Chem 270:10017-10026 (1995); Wondrak, et al., Free Radical Biol. Med. 29:557-567 (2000)). Lander, et al., J. Biol Chem 272: 17810-14 (1997). Interestingly, RCS and AGEs can exert their detrimental cellular effects by increasing ROS production, thereby forming a vicious cycle of ROS and RCS production. AGE-formation is accompanied by accumulation of AGE-specific fluorescence (xcexex-370 nm, xcexem-440 nm) and protein crosslinking, which are measures of overall protein damage in tissue. Brownlee, et al., supra. The arginine-derived imidazolium AGE-products (Westwood, et al., J. Protein Chem 14:359-72 (1995); the glyoxal-lysine dimer (GOLD) and the methylglyoxal-lysine dimer (MOLD) (Brinkmann, et al., J. Biol Chem 273:18714-18719 (1998)) have been identified in aged human lens crystallin and skin collagen implicating xcex1-dicarbonyl stress in tissue aging. Additionally, RCS like glyoxal, the direct precursor of the AGE NE-carboxymethyl-L-lysine (CML), are generated by free radical damage to polyunsaturated fatty acids in cellular membranes. Fu, et al., supra. UV-irradiation is another source of tissue carbonyl stress, as evidenced by the accumulation of CML in sun exposed lesions of actinic elastosis. Mizutani, et al., supra. Therefore, AGE-products like CML and GOLD may be regarded as biomarkers of tissue carbonyl stress.
Methylglyoxal is an important glycation intermediate (Thornalley, et al., supra) that is also generated as a biological metabolite by nonenzymatic and enzymatic degradation of glycolytic triose phosphate intermediates and from threonine catabolism. (Thornalley, et al., Gen. Pharmac 27:565-573 (1996)). Increased levels of methylglyoxal are found in blood from diabetic patients (Beisswenger, et al., Diabetes 48:198-202 (1999)), and in the lens of streptozotocin-induced diabetic rats. A recent study on the formation of AGEs in endothelial cells cultured under hyperglycemic conditions indicated that methylglyoxal was the major precursor of AGEs (Shinohara, et al., J. Clin. Invest. 101:1142-7 (1998)). Various methylglyoxal-derived AGEs have been identified in human tissues, such as fluorescent 5-methylimidazolone-derivatives, in atherosclerotic lesions of aorta (Uchida, et al., FEBS Lett 410:313-318 (1997)), or MOLD and NE-carboxyethyl-L-lysine in aged skin collagen (Brinkmann, et al., supra). Recently, the cytotoxic effects of the glycation intermediates methylglyoxal and 3-deoxyglucosone on neuronal cells such as PC12 cells (Suzuki, et al., J. Biochem (Tokyo) 123:353-7 (1998)) and cultured cortical neurons (Kikuchi, et al., J. Neurosci Res 57:280-289 (1999)) have attracted considerable attention because of their suspected participation in the pathogenesis of neurodegenerative diseases such as Alzheimer""s disease (Vitek, et al., Proc. Natl. Acad Sci USA 91:4766-70 (1994)) and amyotrophic lateral sclerosis (Shinpo, et al., Brain Res. 861:151-9 (2000)).
As another result of oxidative and carbonyl stress, protein damage by carbonylation has been associated with aging and a number of diseases, such as the premature aging diseases, Progeria, and Werner""s syndrome (Berlett, et al., J. Biol Chem 272:20313-20316(1997)). The amount of carbonyl groups in human skin fibroblast proteins strongly correlates with the age of the donor (Oliver, et al., J. Biol Chem 262:5488-5491 (1987). Recently, elevated levels of histone H1 carbonylation in vivo as an indicator of nuclear oxidative and glycoxidative stress have been reported. (Wondrak, et al Biochem J. 351:769-777 (2000)).
In contrast with their therapeutic potential, only a very limited number of biological inhibitors of cellular carbonyl stress, like the nucleophilic carbonyl scavenger glutathione, have been identified to date. However, some inhibitors of glycation interfere with the reaction by trapping intermediate xcex1-dicarbonyls, whereas other inhibitory substances act merely as antioxidants and transition metal chelators, thereby inhibiting advanced glycoxidation, but not glycation (Elgwash, et al., J. Biochem. 271:12964-71 (1996)). Systemic administration of the hydrazine derivative and carbonyl reagent aminoguanidine, a member of the first class of glycation inhibitors, effectively suppresses secondary complications in diabetic rodents with experimental diabetes and inhibits skin collagen crosslinking (Edelstein, et al., Diabetes 41:26-9 (1992); Fu, et al., Diabetes 43: 676-683 (1994)). Recently, a nucleophilic bidentate, phenylacylthiazolium bromide, has been shown to protect E. coli against methylglyoxal toxicity (Ferguson, et al., Chem. Res. Tox 12:617-622 (1999)). Other nucleophilic compounds acting as carbonyl traps like tenilsetam (Shoda, et al., Endocrinol 138:1886-92 (1997)), pyridoxamine (Onorato, et al., J. Biol. Chem 275:21177-21184 (2000)) and metformin (Ruggiero-Lopez, et al., Biochem) are being evaluated for prevention of secondary diabetic complications.
In vitro-screening for potential xcex1-dicarbonyl scavengers is complicated by the nature of most of the currently employed glycoxidative reaction systems, which measure the suppression of oxygen dependent AGE-formation as assessed by AGE-fluorescence or immunological quantification of specific AGEs like CML (Elgawish, et al., supra; Shoda, et al., supra, Ruggiero-Lopez, et al., supra). Consequently, in these glycoxidation systems AGE-formation is effectively inhibited by compounds with antioxidant and metal chelating activity. Recently, oxygen-independent advanced glycation by pentoses with formation of AGE-fluorescence and protein crosslinking has been demonstrated and mechanistically linked to nonoxidative formation of deoxypentosones as reactive xcex1-dicarbonyl intermediates (Litchfield, et al., Int. J. Biochem Cell Biol 31:1297-1305 (1999)).
The foregoing shows that there is an urgent need for the identification and characterization of compounds which effectively inhibit glycation and its associated pathological consequences. Such an assay would be useful in, e.g., analyzing large combinatorial libraries, so as to identify relevant compounds. Such an assay was described in the provisional application referred to supra, and is also described herein. Also see Wondrak, et al., Biochem J. 351:769-777 (2000) incoporated by reference. The work described therein has been pursued further, so as to develop a high throughout screening assay for identifying glycation inhibitors which act as carbonyl scavengers. Prior to these developments, several assay methods had been developed to identify glycation inhibitors for research purposes (Khalifah, et al., Biochem Biophys Res Commun 257:251-258 (1999); Rahbar, et al., Clin. Chem. Acta 287:123-130 (1999); Ruggiero-Lopez, et al., Biochem. Pharmacol 58:1765-1773 (1999)). Parameters that are measured are usually AGE-specific fluorescence, protein cross-linking, and immunological determination of protein-AGE. Most glycation assays employ glucose or a pentose as glycation agent resulting in a slow reaction requiring several weeks for development. These assays typically use non-physiological sugar concentrations or phosphate buffers to increase the rate of the reactions. The sensitivity as measured by level of detection of AGE-formation is low and the precision of the assays is also limited by low signal to noise ratios. The most sensitive assays rely on immunological assessment of AGE-formation by ELISA. Antibodies to AGE used in these assays are either extremely expensive or not commercially available. Hence, it is extremely desirable to have available inexpensive, rapid, easy-to-implement assay for identifying inhibitors of protein glycation, such as inhibitors of non-oxidative protein glycation. It is also desirable to have assays available which mechanistically, do not identify antioxidants, but materials like carbonyl scavengers. Such assays had been developed by the inventors, as will be seen in the description which follows.